Plants the modern vaccines

Presented by Lee-Ann Kara 55103329th April 2014

Introduction

Vaccines

What makes an effective vaccine?

The first Vaccine1885 –Louis Pasteur

http://www.preparedmind.eu/blog/le-hasard-ne-favorise-que-les-esprits-prepares-louis-pasteur/

Delivery systems

Antigen

Immune potentiators

Traditional methods used to produce vaccines

Different types of currently available vaccines:1. Live attenuated vaccines2. Killed vaccines3. Purified subunit vaccines4. Recombinant subunit vaccines.

Recombinant vaccines Advantage* Hepatitis B virus Limitations*

Conventional vaccines: Costly Longer time is needed for it to be produced Heat sensitive Prone to microbial contamination http://www.onlinerevu.com/top-

10-best-paid-to-click-sites/

Biopharming

Two major products in development:1. Plant derived antibodies.2. Plant derived subunit vaccines.

Significance of Plant-derived proteins

First plant derived vaccine

Types of plants as biofactories for vaccine\ antibody production • leaf and stem tissues of tobaccos of various species and varieties (eg:

Nicotiana benthamiana; N tabacum)

• Arabidopsis thaliana,

• Alfalfa,

• Spinach,

• Potatoes; of

• Aquatic weeds suchas Lemna spp. (duckweed);

• Beans

• Maize

• Tomatoes

• Strawberries;

• Root vegetables like carrots;

• single-cell cultures of the algae Chlorella and Chlamydomonas;

Methodologies of vaccine/antibody production

For protein expression:1. Nuclear transformation

Agrobacterium mediated transformation.

Biolistic bombardment transformation.

2. Chloroplast transformation

Biolistic bombardment tranformation

3. Transient expression

Agroinfiltration

Infection with viral vectors

Comparison between protein production systems

Benefits of using transgenic plants

for vaccine production

1. Eliminates possibility of infections or innate toxicity

2. Concerns with viral contamination are eliminated

3. Economical means of large-scale production

4. Maximization of protein stability

5. Reduced cost

6. Multicomponent vaccines are possible

7. Oral administration is possible

SWOT analysis for PDVs

Challenges with PDVs

• Technical challenges

• Economical challenges

• Public perception

Developement of plant- derived vaccines

against cervical cancer

Objectives:

AimTo develop and manufacture a plant-derived vaccine therapeutic against HPV type 16 for females in developing countries which will thereby decrease their risk of developing cervical cancer

To establish a cost-effective expression system for HPV-16 L1

Capsomeres

To express the L1_2xCysM gene that is fused with a adjuvant

LTB in tobacco chloroplasts.

Introduction:

Cervical cancer

~ 493,000 new cases occur every year

To date 274,000 deaths occur in the world

By 2030 it is expected to increase to ~ 410,000

Developing countries have the highest mortality rate due to

cervical cancer is very high

Human Papilloma Virus High-risk types of HPV include HPV-16, 18, 31,

33, 45, 52 and 58.

HPV type 16 and 18

HPV

Circular dsDNA virus

No envelope

L1 protein

Capsid protein self assembles into empty virus

like particles (VLPs)

Modified HPV-16 L1 gene

Disulphide bondshttp://www.virology.wisc.edu/virusworld/images/hpv-HDblue-green.jpg

HPV type 16 will be targeted

Currently available conventional vaccines

Gardasil and Cervarix

Limitations to current available prophylactic vaccines

against HPV infections:• These vaccines target only a limited number of HPV types.

• Expensive due to complex processes of production.

• Separate administration of adjuvant

• Maintenance of cooling chain is costly

Therefore a great demand for cost effective vaccines

for women in poorer countries are needed(Waheed et al., 2012)

Second-generation plant-derived vaccines are the perfect candidates against HPV type 16 infections.

Why? Because of their,

1. Reduced costs

2. Their stability

3. Increased immunogenicity and

4. High yield

Pentameric capsomeres

Capsomeres are found to be highly immunogenic, compared to levels of VLPs.

Major advantages of Capsomeres:

I. Thermo-stable

II. Easy to produce

III. Can be expressed in plants

Modified gene coupled to adjuvants

The modified HPV-16 L1 gene (L1_2xCysM) was expressed in tobacco chloroplasts which led to the formation of only capsomeres.

Tobacco chloroplasts was selected as an expression platform

Reasons for using chloroplasts of Nicotiana tabacum:

Tobbaco is a well established host Tobacco has high Biomass production capacity

Very high yield of recombinant protein can be obtained

Helps assure Biosafety

Rapid scalability

reduced risk of contaminating feed and human food chains

http://www.gizmag.com/darpa-flu-vaccine/25966/pictures

Methodology

Three vectors were constructed for the transformation of tobacco chloroplasts.

Transformation and regeneration of transformed plants

Confirmation of transgenes integration by polymerase chain reaction

Southern blot analysis

Protein extraction

Western blotting

Cesium chloride density gradient centrifugation

Sucrose gradient sedimentation analysis of L1 protein

Confirmation of proper conformation of recombinant proteins

GM1-ganglioside binding assay

http://www.intechopen.com/books/transgenic-plants-advances-and-limitations/green-way-of-biomedicine-how-to-force-plants-to-produce-new-important-proteins

The advantages of plastid transformation method that has been

used which can be exploited for cost effective production of HPV

vaccines:

High yield of recombinant protein can be achieved

Scalability: growth of plants can be scaled up according to the required amount of

protein.

Biosafety issues can be covered by the application of chloroplast transformation and/or

growing the plants in contained facilities.

Transgenic plants can be grown at the site where the vaccine is needed.

Stability: plants-derived vaccines are likely to be more stable.

Commercialization plan Vaccine: Double- pentameric vaccine

Feasibility

• High yields of this vaccine can be produced

• Biosafety

• This vaccine is stable

• 1.5 % - 2% of TSP is accumulated

• Affordable

• Scalability

• Highly effective

Relevance and Importance to society:

• More effective than current available.

• HPV plant vaccines can now be available to people in poorer

countries

Potential Market• Specifically women in developing countries

Transfer technology for commercialization Capital

• Commercialization?? Production costs: Approximately 160 million Rand is needed to

produce 1 billion doses of the Double- pentameric HPV vaccines

Figure 1: showing the difference in production costs between PDVs and conventional vaccines

Time Frames

• Estimated that the double-pentameric HPV vaccine will take 2

weeks to 90 days to be produced in a tobacco plant system

• Egg-based vaccines take 150 days or even longer

Figure 2: Timelines of vaccines produced in different systems

Administration• Subcutaneous injections

Double-pentameric HPV vaccines has a longer shelf life as compared to current available HPV vaccines

References

Ling H-Y, Pelosi A, Walmsley AM (2010) Current status of plantmadevaccines for veterinary purposes. Expert Rev Vaccines9,971–982.Lossl, A.G., and Waheed, M.T. (2011).Chloroplast derived vaccines against human diseases: achievemnents, challenges and scopes. Plant Biotech Journal 9, 527-539. Brodzik R, Spitsin, S., Golovkin, M., Bandurska, K., Portocarrero, C., Okulicz, M., Steplewski, Z.,Koprowski, H. (2008)Plant-derived EpCAM antigen induces protective anti-cancer response. Cancer immunol Immunother 57,317-323. Fernandez-San Millan A, Ortigosa, S. M., Hervas-Stubbs, S., Corral-Martinez, P., Segui-Simarro, J. M., Gaetan, J., Coursaget, P., Veramendi, J.(2008). Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic. Plant Biotechnol J 6,427-441. Varsani A, Williamson, A. L., Stewart, D., Rybicki, E. P.(2006). Transient expression of Humanpapillomavirus type 16 L1 protein in Nicotiana benthamiana using an infectious tobamovirus vector. Virus Res 120,91-96. Aremu AO, Amoo SO, Ndhlala AR, Finnie JF, van SJ. Antioxidant activity, acetylcholinesterase inhibition, iridoid content and mutagenic evaluation of Leucosidea sericea. Food Chem Toxicol 2011;49:1122–8.

Conley, A.J., Joensuu, J.J., Richman, A., Menassa, R. (2011). Protein body-inducing fusions forQ3 high-level production and purification of recombinant proteins in plants. Plant Biotechnol. J . Rybicki EP. Plant-produced vaccines: promise and reality. Drug Discov Today 2009;14: 16–24.Rybicki EP. Plant-made vaccines for humans and animals. Plant Biotechnol J 2010;8:620–37.Rybicki EP, Williamson AL, Meyers A, Hitzeroth II. Vaccine farming in Cape Town. Hum Vaccine 2011;7. Regnard GL, Halley-Stott RP, Tanzer FL, Hitzeroth II, Rybicki EP. High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector. Plant Biotechnol J 2010;8:38–46. Molefe M. Strategy to strengthen the bio-economy, biotechnology landscape in South Africa. National Science and Technology Forum, South Africa, Cape Peninsula University of Technology, Cape Town, South Africa; 2010. p. 27–30Barros E, Nelson SW. Creation of a high-yielding recombinant maize hybrid forthe production of a microbicide to prevent HIV-1 transmission. S Afr J Sci2010;106(5–6):77–81.

Thank You!